Recombinant antibodies against h1n1 influenza

ABSTRACT

Antibodies that bind with high affinity to swine H1N1 virus are described. In vivo experiments showed that one such antibody is able to fully protect mice challenged with a lethal dose of swine H1N1 virus. The antibody is also able to cure mice in a therapeutic setting when treated as late as up to 60 hours (2.5 days) after infection with swine H1N1 virus. Also described are recombinant forms of this antibody.

BACKGROUND

The swine H1N1 influenza virus is currently causing a world-widepandemic associated with substantial morbidity and mortalityl¹⁻⁵. Thisnewly emergent strain is immunologically distinct from other influenzaviruses including recent H1N1 strains⁶ thus leaving a large populationof the world highly susceptible to infection by this pandemic virus⁷.Although there is some B cell cross-reactivity with the seasonalinfluenza viruses the protective epitopes of the swine H1N1 virus appearto be quite distinct.

SUMMARY

Described herein are recombinant antibodies (e.g., human monoclonalantibodies) against the swine H1N1 influenza virus.

Described herein are antibodies derived from plasmablasts isolated frompatients during (or shortly after) infection with the novel influenzavirus. Among the antibodies described herein is an antibody that bindswith particularly high affinity, is highly specific to swine H1N1 virus,and is able to mediate hemagglutination-inhibition at lowconcentrations. In vivo experiments showed that this antibody is able tofully protect mice challenged with a lethal dose of swine H1N1 virus.The antibody is also able to cure mice in a therapeutic setting whentreated as late as up to 60 hours (2.5 days) after infection with swineH1N1 virus. Such antibodies have great potential as a human therapeuticor prophylactic agent against the novel swine H1N1 influenza.

In one aspect, the recombinant antibodies described herein include allor part of the amino acid sequence of SEQ ID NO:1 (light chain) and/orall or part of the amino acid sequence of SEQ ID NO:2 (heavy chain).Within the light chain, the variable domain includes all or part of thesequence of SEQ ID NO:9 and can include one or more of CDR1-light (SEQID NO:3), CDR2-light (SEQ ID NO:4) and CDR3-light (SEQ ID NO:5). Withinthe heavy chain, the variable domain includes all or part of thesequence of SEQ ID NO:10 and can include one or more of CDR1-heavy (SEQID NO:6), CDR2-heavy (SEQ ID NO:7) and CDR3-heavy (SEQ ID NO:8).

Described herein is an isolated antibody or an antigen-binding fragmentthereof that specifically binds the antigen bound by an H1N1 antibodyhaving a light chain consisting of the amino acid sequence of SEQ IDNO:1 and a heavy chain consisting of the amino acid sequence of SEQ IDNO:2. In various embodiments: the antibody or antigen-binding fragmentthereof binds H1N1 (e.g., A/CA/04/2009 H1N1) with a Kd of equal to orless than 10⁻⁹, 10⁻¹⁰ or 6×10⁻¹¹); the antibody or antigen-bindingfragment thereof binds recombinat HA from H1N1 (e.g., A/CA/04/2009 H1N1)with a Kd equal to or less than 10⁻⁹, 10⁻¹⁰ or 9×10⁻¹¹); the antibodycomprises a light chain variable region comprising the amino acidssequences of SEQ ID NOs: 3, 4, and 5; the antibody comprises a heavychain variable region comprising the amino acids sequences of SEQ IDNOs: 6, 7, and 8; the antibody is a human antibody; the antibody is anIgG antibody; the antibody is an IgG1 antibody; the antibody is an IgG1,kappa antibody; the antibody is an IgG1, lambda antibody; the antibodyis selected from an IgM, IgA, IgD and IgE antibody; the antigen-bindingfragment is selected from a Fab, a F(ab′)2 fragment, a Fd fragment, anFv fragment, and a dAb fragment; the antibody is a scFv.

Also described is an isolated antibody or antigen-binding fragmentthereof wherein the antibody comprises: (a) polypeptide comprising theamino acid sequences of one or more of SEQ ID NOs: 3, 4, and 5; and (b)polypeptide comprising the amino acid sequences of one or more of SEQ IDNOs: 6, 7, and 8. In various embodiments: the isolated antibody orantigen-binding fragment thereof comprises: (a) polypeptide comprisingthe amino acid sequences of two or more of SEQ ID NOs: 3, 4, and 5; and(b) polypeptide comprising the amino acid sequences of two or more ofSEQ ID NOs: 6, 7, and 8; the isolated antibody or antigen-bindingfragment thereof comprises: (a) polypeptide comprising the amino acidsequences of SEQ ID NOs: 3, 4, and 5; and (b) polypeptide comprising theamino acid sequences of SEQ ID NOs: 6, 7, and 8; the isolated antibodyor antigen-binding fragment thereof comprises a first polypeptidecomprising, in the amino terminal to carboxy terminal direction aminoacid sequences of two or more of SEQ ID NOs: 3, 4, and 5, wherein thereare 10-20 amino acids between SEQ ID NOs: 3 and 4 and between SEQ IDNOs: 4 and 5; and a second polypeptide comprising, in the amino terminalto carboxy terminal direction amino acid sequences of two or more of SEQID NOs: 6, 7, and 8, wherein there are 10-20 amino acids between SEQ IDNOs: 6 and 7 and between SEQ ID NOs: 7 and 8: the antibody orantigen-binding fragment thereof binds H1N1 (e.g., A/CA/04/2009 H1N1)with a Kd of equal to or less than 10⁻⁹, 10⁻¹⁰ or 6×10⁻¹¹); the antibodyor antigen-binding fragment thereof binds recombinat HA from H1N1 (e.g.,A/CA/04/2009 H1N1) with a Kd equal to or less than 10⁻⁹, 10⁻¹⁰ or9×10⁻¹¹); the antibody comprises a light chain variable regioncomprising the amino acids sequences of SEQ ID NOs: 3, 4, and 5; theantibody comprises a heavy chain variable region comprising the aminoacids sequences of SEQ ID NOs: 6, 7, and 8; the antibody is a humanantibody; the antibody is an IgG antibody; the antibody is an IgG1antibody; the antibody is an IgG1, kappa antibody; the antibody is anIgG1, lambda antibody; the antibody is selected from an IgM, IgA, IgDand IgE antibody; the antigen-binding fragment is selected from a Fab, aF(ab′)2 fragment, a Fd fragment, an Fv fragment, and a dAb fragment; theantibody is a scFv.

Also described is an isolated antibody or antigen-binding fragmentthereof comprising a light chain variable region comprising SEQ ID NOs:3, 4, and 5 and a heavy chain variable region comprising SEQ ID NOs: 6,7, and 8. In various embodiments: In various embodiments: the antibodyor antigen-binding fragment thereof binds H1N1 (e.g., A/CA/04/2009 H1N1)with a Kd of equal to or less than 10⁻⁹, 10⁻¹⁰ or 6×10⁻¹¹); the antibodyor antigen-binding fragment thereof binds recombinat HA from H1N1 (e.g.,A/CA/04/2009 H1N1) with a Kd equal to or less than 10⁻⁹, 10⁻¹⁰ or9×10⁻¹¹); the antibody comprises a light chain variable regioncomprising the amino acids sequences of SEQ ID NOs: 3, 4, and 5; theantibody comprises a heavy chain variable region comprising the aminoacids sequences of SEQ ID NOs: 6, 7, and 8; the antibody is a humanantibody; the antibody is an IgG antibody; the antibody is an IgG1antibody; the antibody is an IgG1, kappa antibody; the antibody is anIgG1, lambda antibody; the antibody is selected from an IgM, IgA, IgDand IgE antibody; the antigen-binding fragment is selected from a Fab, aF(ab′)2 fragment, a Fd fragment, an Fv fragment, and a dAb fragment; theantibody is a scFv.

Also described is a composition comprising an antibody or antigenbinding fragment thereof described herein and a pharmaceuticallyacceptable carrier.

Also described is a method for treating or reducing one or more symptomsof infection with H1N1 in a human subject, the method comprisingadministering an antibody or antigen binding fragment thereof describedherein.

Also described is a method of reducing the risk of becoming infectedwith H1N1, the method comprising administering an antibody describedherein.

Naturally-occurring antibodies are immunoglobulin molecules comprised offour polypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (VH) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region (VL)and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, called complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, called framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

CDRs and FRs may be defined according to Kabat (Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991)). Amino acid numbering of antibodies or antigen bindingfragments is also according to that of Kabat.

Each CDR can included amino acid residues from a complementaritydetermining region as defined by Kabat (i.e. about residues 24-34(CDR-L1), 50-56 (CDR-L2) and 89-97 (CDR-L3) in the light chain variabledomain (SEQ ID NO:1) and 31-35 (CDR-H1), 50-65 (CDR-H2) and 95-102(CDR-H3) in the heavy chain variable domain (SEQ ID NO:2); Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a hypervariable loop (i.e. about residues 26-32(CDR-L1), 50-52 (CDR-L2) and 91-96 (CDR-L3) in the light chain variabledomain (SEQ ID NO:1) and 26-32 (CDR-H1), 53-55 (CDR-H2) and 96-101(CDR-H3) in the heavy chain variable domain (SEQ ID NO:2); Chothia andLesk J. Mol. Biol. 196:901-917 (1987)). In some instances, acomplementarity determining region can include amino acids from both aCDR region defined according to Kabat and a hypervariable loop.

Framework regions are those variable domain residues other than the CDRresidues. Each variable domain typically has four FRs identified as FR1,FR2, FR3 and FR4. If the CDRs are defined according to Kabat, the lightchain FR residues are positioned at about residues 1-23 (LCFR1), 35-49(LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) of SEQ ID NO:1) and the heavychain FR residues are positioned about at residues 1-30 (HCFR1), 36-49(HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) of SEQ ID NO:2. If the CDRscomprise amino acid residues from hypervariable loops, the light chainFR residues are positioned about at residues 1-25 (LCFR1), 33-49(LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain (SEQ IDNO:1) and the heavy chain FR residues are positioned about at residues1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in theheavy chain (SEQ ID NO:2). In some instances, when the CDR comprisesamino acids from both a CDR as defined by Kabat and those of ahypervariable loop, the FR residues will be adjusted accordingly.

An Fv fragment is an antibody fragment which contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in tight association, whichcan be covalent in nature, for example in scFv. It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the VH-VL dimer.Collectively, the six CDRs or a subset thereof confer antigen bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for an antigen) hasthe ability to recognize and bind antigen, although usually at a loweraffinity than the entire binding site.

The Fab fragment contains a variable and constant domain of the lightchain and a variable domain and the first constant domain (CH1) of theheavy chain. F(ab′)2 antibody fragments comprise a pair of Fab fragmentswhich are generally covalently linked near their carboxy termini byhinge cysteines between them. Other chemical couplings of antibodyfragments are also known in the art.

Single-chain Fv or (scFv) antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains, which enables the scFvto form the desired structure for antigen binding.

Diabodies are small antibody fragments with two antigen-binding sites,which fragments comprise a heavy chain variable domain (VH) connected toa light chain variable domain (VL) in the same polypeptide chain (VH andVL). By using a linker that is too short to allow pairing between thetwo domains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites.).

Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1)which, together with complementary light chain polypeptides, form a pairof antigen binding regions. Linear antibodies can be bispecific ormonospecific.

The antibodies herein specifically include chimeric antibodies(immunoglobulins) in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity.

An antigen binding portion of an antibody specifically binds to anantigen (e.g., H1N1). It has been shown that the antigen-bindingfunction of an antibody can be performed by portions of a full-lengthantibody, all of which are encompassed by the general term antibody,including: (i) a Fab fragment, a monovalent fragment consisting of theVL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544 546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423 426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879 5883). Single chain Fv and otherforms of single chain antibodies, such as diabodies are also encompassedby the general term antibody. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl.Acad. Sci. USA 90:6444; Poljak et al. (1994) Structure 2:1121).

An antibody or antigen-binding portion thereof may be part of a largerimmunoadhesion molecules, formed by covalent or noncovalent associationof the antibody or antibody portion with one or more other proteins orpeptides. Examples of such immunoadhesion molecules include use of thestreptavidin core region to make a tetrameric scFv molecule (Kipriyanovet al. (1995) Human Antibodies and Hybridomas 6:93) and use of acysteine residue, a marker peptide and a C-terminal polyhistidine tag tomake bivalent and biotinylated scFv molecules (Kipriyanov et al. (1994)Mol. Immunol. 31:1047). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques.

Human antibodies include antibodies having variable and constant regionsderived from (or having the same amino acid sequence as those derivedfrom) human germline immunoglobulin sequences. Human antibodies mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3.

Recombinant antibodies are prepared, expressed, created or isolated byrecombinant means, such as antibodies expressed using a recombinantexpression vector transfected into a host cell, antibodies isolated froma recombinant, combinatorial human antibody library, antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (Taylor et al. (1992) Nucl. Acids Res. 20:6287) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences orvariants thereof to other DNA sequences. Such recombinant humanantibodies have variable and constant regions derived from humangermline immunoglobulin sequences or variants thereof. In certainembodiments, however, such recombinant human antibodies are subjected toin vitro mutagenesis (or, when an animal transgenic for human Igsequences is used, in vivo somatic mutagenesis) and thus the amino acidsequences of the VH and VL regions of the recombinant antibodies aresequences that may not naturally exist within the human antibodygermline repertoire in vivo.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Generation of human monoclonal antibodies against swine H1N1influenza virus from plasmablasts of infected patients. (a)Antibody-secreting B cells in the PBMC of swine influenza virus infectedpatients were isolated by flow cytometry sorting based on their cellsurface phenotype (CD19⁺, CD20⁻, CD3⁻, CD38^(high) and CD27^(high)).RT-PCR was used to isolate the variable genes from sorted singleplasmablasts, which were then cloned into expression vectors andexpressed in 293 cells as we have previously described^(11, 12). (b)Forty-seven percent ( 25/53) of the monoclonal antibodies generatedbound to purified swine H1N1 (A/CA/04/2009) virus as determined byELISA. (c) Five of 53 antibodies bound to recombinant swine H1N1hemagglutinin (rHA), but only one of these mAbs (EM4C04) could inhibithemagglutination (HAI+) of erythrocytes by the swine H1N1 influenzastrain (d).

FIG. 2. A majority of the antibodies induced by swine H1N1 infection arecrossreactive to seasonal influenza strains. Antibodies generated duringactive infection with the swine H1N1 strain (top line) were screened byELISA for reactivity to various influenza antigens (indicated within thefigure). Bars indicate the area under the curve, thus providing insightinto both the maximal binding (Bmax) and persistence of binding withdecreasing dilutions (affinity or Kd). Note that only a few antibodieswere specific just to the swine H1N1 strain alone and that a number ofantibodies bound to annual influenza vaccine strains either solely orwith higher affinity (indicated with asterisks). In total 47% ( 25/52)bound swine H1N1 and 58% ( 30/52) bound influenza antigens at levelsdetectable by ELISA assay¹². The mAb EM4C04 (bold) had the highest andmost specific affinity against swine H1N1. Cocktail: A/Sal. Is./3/2006(H1N1), A/WI/57/05 (H3N2), and B/Mal./2506/04, 2006/7 Vaccine: A/NewCal./20/90 (H1N1), A/WI/57/05 (H3N2), and B/Mal./2506/04, 2008/9Vaccine: A/Brisb./59/2007 (H1N1), A/Brisb./10/2007 (H3N2),andB/FL/4/2006.

FIG. 3. The monoclonal antibody EM4C04 is highly specific for the swineH1N1 influenza hemagglutinin and displays HAI activity only to the swineH1N1 virus. (a) ELISA binding curves of the mAb EM4C04, comparingbinding to whole virus with reactivity to viral mixtures or the annualvaccines as indicated, to purified virions or to recombinanthemagglutinin (rHA) from swine H1N1 versus other influenza strains.Calculated Kd values are shown in parenthesis above the graphs.Cocktail: A/Sal.Is./3/2006 (H1N1), A/WI/57/05 (H3N2), andB/Mal./2506/04, 2006/7 Vaccine: A/New Cal./20/90 (H1N1), A/WI/57/05(H3N2), and B/Mal./2506/04, 2008/9 Vaccine: A/Brisb./59/2007 (H1N1),A/Brisb./10/2007 (H3N2),and B/FL/4/2006. (b) EM4C04 is able toimmuno-precipitate recombinant from swine H1N1 HA protein. (c) EM4C04displays HAI activity toward swine H1N1 but not to several other H1N1strains tested as indicated.

FIG. 4. EM4C04 has therapeutic efficacy in mice challenged with a lethaldose of mouse-adapted 2009 swine H1N1 influenza. 6-8 week old Balb/cmice were infected with a 3×LD50 dose of highly pathogenic,mouse-adapted 2009 swine H1N1 influenza (A/California/04/09).Subsequently, they were treated with 200 mg (10 mg/kg of body weight)EM4C04 human monoclonal antibody intraperitoneally at various timepoints (12, 24, 36, 48 and 60 hours) after infection. All mice weremonitored daily for body weight changes and any signs of morbidity andmortality. Infected, untreated mice showed clear signs of sicknessaround day 4-5 post infection and perished by day 8-9. Upper panels showbody weight change and the lower panels show survival curves.

FIG. 5. Plasmablasts expressing antibodies that cross-react to annualinfluenza strains have accumulated more somatic hypermutations. Thehigher frequency of mutations in the more crossreactive antibodiesindicate that they were derived from a recall response of memory Bcells, originally induced by annual influenza viruses. It is alsonotable that a number of IgG+ plasmablasts that had no detectablebinding to influenza by ELISA were from cells that had no mutations ofthe variable genes. The origin and specificity of these cells is unknownbut they may be cells activated during a primary response against swineH1N1 epitopes that had affinities below the threshold of detection. Thefrequency of point mutations was determined from the variable genesequences of the VH and Vκ sequences that were generated for the cloningand expression of the antibodies. Points represent the sum of heavy andlight chain mutations. Statistical significance was determined bystudents t test.

FIG. 6. Prophylactic treatment with EM4C04 can protect mice from alethal challenge with mouse-adapted swine H1N1 influenza. 6-8 week oldBalb/c mice were treated with 200 μg (10 mg/kg of body weight) EM4C04human monoclonal antibody intraperitoneally 12 hours prior to infectionwith a 3×LD50 dose of highly pathogenic mouse adapted swine H1N1influenza. All mice were monitored daily for body weight changes and anysigns of morbidity and mortality. Upper panels show body weight changeand the lower panels show survival curves.

DETAILED DESCRIPTION

The studies described below analyzed the B cell responses in patientsinfected with swine H1N1 virus. As part of these studies we generated apanel of virus specific human monoclonal antibodies. These antibodieswere isolated from plasmablasts that were activated by infectionproviding a means to directly evaluate the breadth and repertoire of theantibody response elicited by swine H1N1 virus. Interestingly, amajority of these antibodies also reacted with seasonal influenzaviruses. In fact, several of the antibodies bound with higher affinityto past influenza strains than to the current swine H1N1 virus. Thesefindings suggest that the swine H1N1 virus predominantly activatedmemory B cells previously generated against cross-reactive butnon-protective epitopes present in annual influenza strains. Of theinfluenza specific antibodies generated five bound to recombinanthemagglutinin (HA) protein and of these only one antibody showedhemagglutination-inhibition (HAI) activity against the swine H1N1influenza virus. In contrast to most of the other antibodies generated,this neutralizing antibody was highly specific for the swine H1N1 virusand did not cross-react with the other H1N1 influenza viruses,confirming that the critical HA active-site epitopes in this new virusare quite unique. In vivo experiments showed that this antibody was ableto protect mice challenged with a lethal dose of mouse-adapted swineH1N1 influenza virus. Moreover, it was effective therapeutically evenwhen administered 60 hours after infection and could thus potentially bedeveloped as a therapeutic agent against the swine H1N1 influenza viruspandemic.

The novel 2009 pandemic swine H1N1 influenza virus is characterized by aunique genetic make-up^(1,2,8) that results in little or no pre-existingserum antibody mediated protection against infection^(7,9). It iscurrently unclear what effect this has on the repertoire of responding Bcells in infected patients and whether infection with this novel virusleads to activation of cross-reactive memory B cells or if the responseis dominated by newly induced naive B cells. To analyze the repertoireof the responding B cells after infection and to generate monoclonalantibodies (mAbs) against the swine H1N1 influenza strain, we examinedthe B cell responses in five patients infected with swine H1N1 virus.The clinical details about these patients are given in the supplementalmethods section. Blood samples were taken 1-2 weeks after onset ofclinical symptoms and were used to isolate infection-inducedplasmablasts (CD19⁺, CD20⁻, CD3⁻, CD38^(high) and CD27^(high) cells) byflow cytometry based cell sorting (FIG. 1 a shows a representativedonor). Using an adapted single cell multiplex RT-PCRapproach^(10,11,12,) we then identified the heavy and light chainimmunoglobulin genes from each individual plasmablast from two of thefive patients. These heavy and light chain fragment pairs were then usedto express fully human monoclonal antibodies. In total, 25 out of the 53(47%) antibodies generated in this fashion bound to purified whole swineH1N1 influenza (A/CA/04/2009) virus by ELISA (FIG. 1 b). It is notablethat the majority of antibodies induced by infection were low affinity;only five of the 53 isolated antibodies, had affinities >¹⁰⁻⁹ bynon-linear regression analysis of ELISA data. Further, as indicated inFIG. 1 c, five of the 53 antibodies bound to recombinant hemagglutinin(rHA) from swine H1N1 influenza by ELISA, but only one of these mAbs(EM4C04) displayed HAI activity (FIG. 1 d). We conclude from thisanalysis that a large proportion of virus specific plasmablasts in thesepatients were not producing neutralizing antibodies, and that themajority of the B cell response was in fact directed at non-HA proteins.

In order to determine how specific the antibody response was to theswine H1N1 virus strain, the 53 monoclonal antibodies were screened byELISA for reactivity to various influenza antigens (FIG. 2). The bars inFIG. 2 indicate the area under the curve of ELISA binding data (FIG. 1a), thus providing an overview of both the maximal binding (B_(max)) andthe persistence of binding with decreasing dilutions (affinity orK_(d)), allowing a relative comparison of each antibody to all antigensby column. It is notable that most of the antibodies were indeedcross-reactive with past strains of influenza virus, suggesting thatthey arose through the activation of cross-reactive memory B cells. Intotal, 47% ( 25/52) of the antibodies bound to swine H1N1 and 58% (30/52) bound to antigens from any of the influenza strains tested. Infact, 23% of the antibodies bound to past annual influenza strains withhigher affinity than to the swine H1N1 strain (FIG. 2, asterisks). Theplasmablasts expressing antibodies that were cross-reactive to pastannual influenza strains had also accumulated significantly moremutations in the variable genes on average than the swine H1N1-specificB cells (FIG. 5). These findings suggest that the swine H1N1 strainpredominantly activated memory B cells previously generated againstcross-reactive but non-protective epitopes present in annual influenzavirus strains.

It is worth noting that the sole HAI⁺mAb, EM4C04 (FIG. 2) was also themost specific against swine H1N1, demonstrating that the critical HAactive-site epitopes are quite unique, as predicted by analyses of theHA amino acid sequences by several other groups^(1,2,7,13). The highspecificity of EM4C04 demonstrates that this antibody could be valuablefor diagnostic purposes for the pandemic swine H1N1 influenza virus(FIG. 3 a). This antibody was also able to immuno-precipitaterecombinant HA protein derived from swine H1N1 (FIG. 3 b). In addition,while EM4C04 efficiently inhibited the agglutination of red blood cellsby swine H1N1 virus, it had no HAI activity against several otherinfluenza strains (FIG. 3 c). The high affinity (6.1x¹⁰⁻¹¹ to purifiedvirus and 9x¹⁰⁻¹¹ to rHA) for the HA active site suggested that thisantibody could be used for passive immunization to treat swine H1N1influenza infection. We therefore tested the prophylactic andtherapeutic potential of EM4C04 in mice infected with a lethal dosage ofhighly pathogenic, mouse-adapted swine H1N1 strain.

As indicated in FIG. 4 (and FIG. 6), the EM4C04 antibody is highlyeffective at either providing prophylactic protection against infectionor to treat and facilitate clearance of a lethal dose of mouse-adaptedswine H1N1 from 6-8 week old Balb/c mice. For the prophylacticexperiments mice were pretreated with 200 μg EM4C04 human monoclonalantibody intraperitoneally and then challenged 12 hours later with a3×LD50 dose of mouse-adapted novel H1N1 influenza (FIG. 6). To determinethe therapeutic potential of EM4C04, mice were first challenged and thentreated with antibody at various times after infection (FIG. 4). Whileuntreated mice died 8-9 days after the infection, mice treated even aslate as 60 hours after challenge survived. Infected mice treated atlater time points were already showing measurable weight loss that wasreversed by administration of the antibody, demonstrating therapeuticpotential even after the onset of symptoms. Overall, 30 of 31 infectedmice that were treated with EM4C04, irrespective of when they weretreated, made a complete recovery from infection. It is likely that thetherapeutic effects of EM4C04 treatment involve both direct viralneutralization as well as facilitation of endogenous cell-mediatedimmunity¹⁴. It is possible that the antibody treatment may reduce viraltiters and thus allow the endogenous immune responses to catch up andsubsequently clear the infection.

The studies show that the antibody responses induced in patientsinfected with the novel swine H1N1 influenza appear to be dominated by arecall response of non-protective memory B cells that are cross-reactiveto annual influenza strains. Of the 25 virus-specific monoclonalantibodies generated herein only one displayed HAI activity against theswine H1N1 virus. This low frequency of cells producing protectiveantibodies after infection differs significantly as compared to previouswork on seasonal influenza vaccines¹², where 40% of the virus specificantibodies bound with high affinity to HA and half of those antibodieshad HAI activity against the influenza vaccine viral strains. As thenovel swine H1N1 vaccine is now becoming widely available¹⁵⁻¹⁸, it willbe of interest to compare the vaccine induced antibody responses to theresponses induced by infection as described herein. Finally, the in vivoprotection experiments presented here demonstrate that the humanmonoclonal antibody EM4C04 has impressive prophylactic and therapeuticactivity in mice and shows potential for development as a therapeuticagent against the pandemic swine H1N1 influenza virus in humans.

METHODS

Patients were recruited with IRB approval and had ongoing or recentverified swine H1N1 infections. HAI titers, inhibiting antibodyconcentrations, and viral neutralization were determined by standardprocedures as previously described^(12,19). The ASCs were identifiedherein as CD3⁻/CD20⁻/^(low)/CD19⁺/CD27^(hi)/CD38^(hi) cells aspreviously described^(11,12). The single cell RT-PCR methods and theprocedures for production of recombinant mAbs were as previouslydescribee¹⁰⁻¹². Monoclonal antibodies were screened against freshinfluenza virions grown in chicken eggs. ELISA was performed on startingconcentrations of 10 ug/ml of virus or rHA and on 1:20 dilution of thevaccines and antibody affinities (Kd) were calculated by nonlinearregression analysis as previously described¹². For immunoprecipitation,1 μg each of recombinant HA protein and antibody were incubated at 4° C.overnight in 100 μl NP40 Buffer prior to precipitation with ProteinG-Sepharose. The samples were denatured for 5 min at 95° C. in Laemmligel sample buffer followed by centrifugation to remove the ProteinGSepharose and analysis on 12% Tris-Glycine polyacrylamide gels.Precipitated protein bands were identified by staining with Sypro-orangeand Fluorescence imaging. For the challenge experiments, female Balb/cmice (8 weeks old) were challenged intra-nasally with 3×xLD₅₀ of ahighly pathogenic, mouse-adapted swine H1N1 influenza virus(A/California/04/09) that was passaged in mice for five generations.Mice were treated intraperitoneally with 200 ug (10 mg/kg of bodyweight) of the specific mAb EM4C04 at all time points. All mice weremonitored daily for morbidity and body weight changes.

Patients

All studies were approved by the Emory University, University of Chicagoand Columbia University institutional review boards (Emory IRB#22371 and555-2000, U of C IRB# 16851E, CU IRB#AAAE1819). Patient 1 (EM) is a30-year old healthy woman who developed fever, cough and progressivedyspnea over 8 days prior to hospital admission. She was diagnosed withacute respiratory syndrome (ARDS), which required mechanicalventilation. Her nasopharyngeal swab on admission was positive forinfluenza by RTPCR. She continued shedding virus (hospital day 13)despite treatment with oseltamivir, but had cleared the virus by day 15with continued treatment. Her course was further complicated bybacterial pneumonia, pulmonary embolism, and a requirement for prolongedoscillatory ventilator support and tracheostomy. She gradually recoveredand was discharged to home two months after becoming ill. Blood samplesfor PBMC preparation were collected 19 days and 29 days after the onsetof symptoms. Patient 2 (SF) is a 37-year old man with a history ofhypertension and interstitial lung disease of unknown etiology who washospitalized with symptoms of fever, cough, shortness of breath, nauseaand vomiting for 3 days. He was diagnosed with pneumonia, acutesinusitis and acute renal failure. His nasopharyngeal swab on admissionwas positive for influenza virus by culture and was confirmed as theswine H1N1 influenza virus by RTPCR. He was initially treated withoseltamivir for 5 days but was continuing to shed influenza virus andwas discharged with a course of zanamivir. He was hospitalized for atotal of 8 days and recovered. PBMCs were collected 18 days after theonset of symptoms. Patient 3 is a 25 year old male who developed coughand fever to 103° F. The diagnosis of 2009 H1N1 influenza was confirmedby RT-PCR. He was treated with oseltamivir and his symptoms lasted for 4days. He recovered completely and blood samples were collected 9 daysafter the onset of symptoms. Patient 4 is a previously healthy, 40-yearold man who developed symptoms consistent with mild upper respiratorytract illness, including cough, rhinorrhea, and fever. MassTag PCRanalysis of a nasopharyngeal swab specimen obtained 6 days after symptomonset identified H1N1 influenza virus; the presence of swine H1N1influenza virus was subsequently confirmed by RT-PCR. Blood samples forPBMC isolation were obtained 13 days after the onset of symptoms.Patient 5 is a 52 year old female whose diagnosis of 2009 H1N1 influenzaA was confirmed by RT-PCR. Her symptoms included fever, cough,pharyngitis, myalgias, nausea, headache, and gastrointestinal symptoms.She was treated with oseltamivir and her symptoms resolved after 6 daysand she recovered completely. Blood samples were collected 10 days afterthe onset of symptoms.

Cell and Serum Isolation

All work with samples from infected patients was performed in adesignated BSL2+ facility at Emory University. Peripheral bloodmononuclear cells (PBMC) were isolated using Vacutainer tubes (BectonDickinson, BD), washed, and resuspended in PBS with 2% FCS for immediateuse or frozen for subsequent analysis. Plasma samples were saved in−80C.

Viruses and Antigens

The Swine H1N1 influenza virus (A/California/04/2009) was kindlyprovided by Dr. Richard J Webby at St. Jude Childrens Hospital.Influenza virus stocks used for the assays were freshly grown in eggs,prepared and purified as described¹⁹ and the hemagglutination activity(HA) was determined using turkey red blood cells (Lampire BiologicalLaboratories, Pipersville, Pa.) as previously described^(12,19) orpurchased as inactivated preparations (ProSpec-Tany TechnoGene Ltd.,Rehovot, Israel) and included: A/California/04/2009 (H1N1), A/FM/1/47(H1N1), A/PR8/34 (H1N1), A/New Jersey/76 (H1N1), A/New Caledonia/20/9(H1N1), A/Solomon Island/3/2006, A/Wisconsin/67/2005 (H3N2), andB/Malaysia/2506/2004. Vaccines tested included the 2006/7 vaccine fromChiron Vaccines Limited (Liverpool, UK) and the 2008/9 formulation fromSanofi Pasteur Inc. (Swiftwater, Pa.). Recombinant HA proteins wereprovided by the influenza reagent resource (IRR; influenza reagentresource.org) of the CDC (rHA from A/California/04/2009 (H1N1)(#FR-180), A/Brisbane/10/2007 (H1N1) (#FR-61), A/Brisbane/59/2007 (H3N2)(#FR-65)) or by Biodefense & Emerging Infections research repository(BEI; www.beiresources.org) (rHA from A/Indonesia/05/2005).

Flow Cytometry Analysis and Cell Sorting

Analytical flow cytometry analysis was performed on whole bloodfollowing lysis of erythrocytes and fixing in 2% PFA. All live cellsorting was performed on purifiedPBMCs in the BSL-3 facility at theEmory Vaccine Center. All antibodies for bothanalytical and cell sortingcytometry were purchased from Pharmingen, except anti-CD27 that waspurchased from ebiosciences. Anti-CD3-PECy7 or PerCP, anti-CD20-PECy7 orPerCP, anti-CD38-PE, anti-CD27-APC and anti-CD19-FITC. ASCs were gatedand isolated as CD19⁺CD3⁻CD20^(low)CD27^(high)CD38^(high) cells. Flowcytometry data was analyzed using FlowJo software.

Generation of Monoclonal Antibodies

Identification of antibody variable region genes were done essentiallyas previously described^(10,11). Briefly, single ASCs were sorted into96-well PCR plates containing RNase inhibitor (Promega). VH and Vicgenes from each cell were amplified by RT-PCR and nested PCR reactionsusing cocktails of primers specific for both IgG and IgA as previouslydescribee^(10,11) and then sequenced. To generate recombinantantibodies, restriction sites were incorporated by PCR with primers tothe particular variable and junctional genes. VH or Vκ genes amplifiedfrom each single cell were cloned into IgG1 or Igκ expression vectors aspreviously describee^(10,11). Heavy/light chain plasmids wereco-transfected into the 293A cell line for expression and antibodiespurified with protein A sepharose.

ELISA and HAI Assays

Whole virus, recombinant HA or vaccine-specific ELISA was performed onstarting concentrations of 10 ug/ml of virus or rHA and on 1:20 dilutionof the vaccine as previously described¹². The hemagglutinationinhibition (HAI) titers were determined as previously described^(11,19).Affinity estimates were calculated by nonlinear regression analysis ofcurves from 8 dilutions of antibody (10 to 0.125 μg/ml) using GraphPadPrism.

Immunoprecipitation

For immunoprecipitation, 100 μl NP40 Buffer (20 mM Tris-HCl PH8.0, 137mM NaCl, 10% Glycerol, 1% NP-40, 2 mM EDTA) containing complete ProteaseInhibitors (Roche) was mixed with 1 μg of recombinant HA protein andincubated on ice for 30 min. One microgram of monoclonal antibody wasthen added. The antibody and HA mixture was incubated at 4° C. overnightwith constant agitation. On the next day, Protein G-Sepharose (GEHealthcare) was prepared in NP40 buffer at a volume of 10 μl/sample.Protein GSepharose was incubated with the antibody and HA mixture at 4Cfor 4 hrs with constant agitation. The protein G-Sepharose wascentrifuged for 3 min at 3000 rpm and the pellet was washed with 400 μlof NP40 buffer for 3 times. Finally the pellet was resuspended into 25μl of Laemmli gel sample buffer (Bio-Rad). The samples were then boiledfor 5 min at 95C. The protein G was pelleted and 15 μl of supernatantwas loaded onto 12%Tris-Glycine polyacrylamide gels. The gels were runin 1×TGS at 70V for 30 min, followed by 120V till the frontline ran outof the gel. The gels were stained with 1× Sypro-orange (Invitrogen) in7.5% acetic acid for 1 hr, and then gels were destained with 7.5% aceticacid for 3 min. Gels were finally scanned in a Typhoon 9410 Fluorescenceimaging system (GE Healthcare).

In vivo Protection Experiments

Female Balb/c mice 6-8 weeks old were used for the challenge studies.Mice were inoculated intra-nasally with 3xLD₅₀ of a highly pathogenic,mouse-adapted swine H1N1 influenza virus (A/California/04/09) that waspassaged in mice five generations. The LD₅₀ was determined by the methodof Reed and Muench. The experiments were conducted in accordance withethical procedures and policies approved by the Emory University'sInstitutional Animal Care and Use Committee. In order to determine theprophylactic efficacy of the mAb, mice were treated intraperitoneallywith 200 μg (10 mg/kg of body weight) of the specific mAb EM4C04. Twelvehours later mice were challenged with 3xLD₅₀ of the mouse adapted H1N1virus. All mice were monitored daily for any signs of morbidity andmortality. Body weight changes were registered daily for a period of 14days. All mice that lost more that 25% of their initial body weight weresacrificed according to the IACUC guideless. In order to determine thetherapeutic efficacy of the EM4C04 mAb, mice were challenged with 3xLD₅₀of the mouse-adapted swine H1N1 virus. At various times post infection(12, 24, 36, 48, 60 hours) mice were treated intraperitoneally with 200μg (10 mg/kg of body weight) of the specific mAb EM4C04. All mice weremonitored daily and the body weight changes were registered daily asdescribed above.

Statistical Analysis

Data was collected and graphed using MS Excel and Graphpad Prismsoftware. Efficacy of the therapeutic and challenge experiments wasevaluated by ANOVA using Graphpad Prism software.

Sequences of Antibodies

Described below are the sequences of the EM4C04 heavy chain and lightchain

EM4C04 Heavy Chain Variable Region: DNAGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTTCAGCCTCTGGATTCACCTTCAATATCTATGCCATGAACTGGGTCCGCCAGGTTCCAGGAAAGGGGCTGGATTGGGTCTCATCCATTAGTAGTAGGGGTGATTACATATACTACGCAGAGTCAGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGGAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGCTGGGCTGGGTACAGTGGATTTAAGGTGGGGGGGGGCCTTCGACCACTGGGGCAAGGGAATCCTGGTCAC CGTCTCCTCAAmino Acid: (SEQ ID NO: 2)EVQLVESGGGLVKPGGSLRLSCSASGFTFNIYAMNWVRQVPGKGLDWVSSISSRGDYIYYAESVEGRFTISRDNAKNSLYLEMNSLRAEDTAVYYCARAGLGTVDLR WGGAFDHWGKGILVTVSSAlignment: Ig Sequence  EM-Swinel-4C04H- Name: V gene:Z14073_IGHV3-21*01 D Gene: None Found D Gene 2: None Found J Gene:X86355 IGHJ5*02 Clonal Pool: 0 CDR3 Length: 17 CDR3 AA:RAGLGTVDLRWGGAFDH 1> Z14073_1GHV3-21*01    20           30            40 E   V   Q   L   V   E   S   G   G   G   L   V   K   P   G GermlineGAG GTG CAG CTG GTG GAG TCT GGG GGA GGC CTG GTC AAG CCT GGGEM-Swine1-4C04H---- --- --- --- --- --- --- --- --- --- --- --- --- --- ---     50           60            70           80          90 G   S   L   R   L   S   C   A   A   S   G   F   T   F   S GermlineGGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC TTC AGTEM-Swine1-4C04H---- --- --- --- --- --- --- T-- --- --- --- --- --- --- -A-                             S                           N            100          110          120           130 S   Y   S   M   N   W   V   R   Q   A   P   G   K   G   L GermlineAGC TAT AGC ATG AAC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTGEM-Swine1-4C04H--T- --- GC- --- --- --- --- --- --- -T- --- --A --- --- --- I       A                           V     140          150           160          170         18 E   W   V   S   S   I   S   S   S   S   S   Y   I   Y   Y GermlineGAG TGG GTC TCA TCC ATT AGT AGT AGT AGT AGT TAC ATA TAC TACEM-Swine1-4C04H---T --- --- --- --- --- --- --- --G G-- GA- --- --- --- --- D                               R   G   D0           190          200          210           220 A   D   S   V   K   G   R   F   T   I   S   R   D   N   A GermlineGCA GAC TCA GTG AAG GGC CGA TTC ACC ATC TCC AGA GAC AAC GCCEM-Swine1-4C04H---- --G --- --- G-- --- --- --- --- --- --- --- --- --- ---     E           E     230          240           250          260         27 K   N   S   L   Y   L   Q   M   N   S   L   R   A   E   D GermlineAAG AAC TCA CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GACEM-Swine1-4C04H---- --- --- --- --- --- G-- --- --- --- --- --- --- --- ---                         E0           280          290          300           310 T   A   V   Y   Y   C   A   ?   ?   ?   ?   ?   ?   ?   ? GermlineACG GCT GTG TAT TAC TGT GCG AGN NNN NNN NNN NNN NNN NNN NNNEM-Swine1-4C04H---- --- --- --- --- --- --- --A GCT GGG CTG GGT ACA GTG GAT                             R   A   G   L   G   T   V   D     320          330   1> X86355 IGHJ5*02   350         36  ?   ?   ?  ?   ?   ?   F   D   P   W   G   Q   G   T   L GermlineNNN NNN NNN NNN NNN NNN TTC GAC CCC TGG GGC CAG GGA ACC CTGEM-Swine1-4C04H-TTA AGG TGG GGG GGG GCC --- --- -A- --- --- A-- --- -T- --- L   R   W   G   G   A          H            K       I 0           370 V   T   V   S   S   ? Germline GTC ACC GTC TCC TCA G EM-Swine1-4C04H---- --- --- --- --- - EM4C04 kappa Variable Domain: DNAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCTCTTGCCAGGCGAGTCAGGATATTACCAACTTTTTAAATTGGTACCAGCAGAAATCTGGGGAAGCCCCTAAGCTCCTGATCTACGATGCATCCGATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGGCTGCAGCCTGAAGACACTGCAACATATTACTGTCAACAGTATGACGATCTCCCGTATACTTTTGGCCAGGGGACCAAGGTGGAG ATCAAA Amino acid(SEQ ID NO: 1) DIQMTQSPSSLSASVGDRVTISCQASQDITNFLNWYQQKSGEAPKLLIYDASDLETGVPSRFSGSGSGTDFTFTISRLQPEDTATYYCQQYDDLPYTFGQGTKVEIK Alignment:Ig Sequence  EM-Swine1-4C04K- Name: V gene: M64855_IGKV1D-33*01 D Gene:None Found D Gene 2: None Found J Gene: J00242 IGKJ2*01 Clonal Pool: 0CDR3 Length: 8 CDR3 AA: QYDDLPYT 1>M64855_IGKV1D-33*01   20           30            40 D   I   Q   M   T   Q   S   P   S   S   L   S   A   S   V GermlineGAC ATC CAG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCA TCT GTAEM-Swine1-4C04K---- --- --- --- --- --- --- --- --- --- --- --- --- --- --T     50           60            70           80          90 G   D   R   V   T   I   T   C   Q   A   S   Q   D   I   S GermlineGGA GAC AGA GTC ACC ATC ACT TGC CAG GCG AGT CAG GAC ATT AGCEM-Swine1-4C04K---- --- --- --- --- --- T-- --- --- --- --- --- --T --- -C-                         S                               T            100          110          120           130 N   Y   L   N   W   Y   Q   Q   K   P   G   K   A   P   K GermlineAAC TAT TTA AAT TGG TAT CAG CAG AAA CCA GGG AAA GCC CCT AAGEM-Swine1-4C04K---- -T- --- --- --- --C --- --- --- T-T --- G-- --- --- ---     F                               S       E     140          150           160          170         18 L   L   I   Y   D   A   S   N   L   E   T   G   V   P   S GermlineCTC CTG ATC TAC GAT GCA TCC AAT TTG GAA ACA GGG GTC CCA TCAEM-Swine1-4C04K---- --- --- --- --- --- --- G-- --- --- --- --- --- --- ---                             D0           190          200          210           220 R   F   S   G   S   G   S   G   T   D   F   T   F   T   I GermlineAGG TTC AGT GGA AGT GGA TCT GGG ACA GAT TTT ACT TTC ACC ATCEM-Swine1-4C04K---- --- --- --- --- --- --- --- --- --- --- --- --- --- ---     230          240           250          260         27 S   S   L   Q   P   E   D   I   A   T   Y   Y   C   Q   Q GermlineAGC AGC CTG CAG CCT GAA GAT ATT GCA ACA TAT TAC TGT CAA CAGEM-Swine1-4C04K---- --G --- --- --- --- --C -C- --- --- --- --- --- --- ---     R                       T 0           280         1>J00242 IGKJ2*01          310  Y   D   N   L   ?   ?  T   F   G   Q   G   T   K   L   E GermlineTAT GAT AAT CTC CCN NNN ACT TTT GGC CAG GGG ACC AAG CTG GAGEM-Swine1-4C04K---- --C G-- --- --G TAT --- --- --- --- --- --- --- G-- ---         D       P   Y                               V      320 I   K   ? Germline ATC AAA C EM-Swine1-4C04K- --- --- -CDR and FR of EM4C04 Heavy Chain: Nucleotide: FW1:GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTTCAGCCTCTGGATTCACCTTCAAT CDR1: ATCTATGCCATGAAC FW2:TGGGTCCGCCAGGTTCCAGGAAAGGGGCTGGATTGGGTCTCA CDR2:TCCATTAGTAGTAGGGGTGATTACATATACTACGCAGAGTCAGTGGAGGGC FW3:CGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGGAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGA CDR3:GCTGGGCTGGGTACAGTGGATTTAAGGTGGGGGGGGGCCTTCGACCAC FW4:TGGGGCAAGGGAATCCTGGTCACCGTCTCCTCA Amino Acids: FW1:EVQLVESGGGLVKPGGSLRLSCSASGFTFN CDR1: (SEQ ID NO: 4) IYAMN FW2:WVRQVPGKGLDWVS CDR2: (SEQ ID NO: 5) SISSRGDYIYYAESVEG FW3:RFTISRDNAKNSLYLEMNSLRAEDTAVYYCAR CDR3: AGLGTVDLRWGGAFDH FW4:(SEQ ID NO: 6) WGKGILVTVSS CDR and FR of EM4C04 Light Chain: Nucleotide:FW1: GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCTCTTGCCAGGCGAGT CDR1: CAGGATATTACCAACTTTTTAAAT FW2:TGGTACCAGCAGAAATCTGGGGAAGCCCCTAAGCTCCTGATCTAC CDR2:GATGCATCCGATTTGGAAACA FW3:GGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGGCTGCAGCCTGAAGACACTGCAACATATTACTGT CDR3:CAACAGTATGACGATCTCCCGTATACT FW4: TTTGGCCAGGGGACCAAGGTGGAGATCAAAAmino acids: FW1: DIQMTQSPSSLSASVGDRVTISC CDR1: (SEQ ID NO: 3)QASQDITNFLN FW2: WYQQKSGEAPKLLIY CDR2: (SEQ ID NO: 4) DASDLET FW3:GVPSRFSGSGSGTDFTFTISRLQPEDTATYYC CDR3: (SEQ ID NO: 5) QQYDDLPYT FW4:FGQGTKVEIK

The CDR described herein can be grafted into the following vectorsencoding human IgG and kappa chains, as well as others: Fully human IgG(GenBank® Accession No: FJ475055) and Fully human kappa (GenBank®Accession No: FJ475056).

GenBank ® FJ475055RSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK    1ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca   61ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct  121ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta  181acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac  241ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt  301aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag  361tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat  421gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat  481gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc  541ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt  601ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga  661caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt  721gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta  781gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac  841tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc  901acctcggttc tatcgattga attccaccat gggatggtca tgtatcatcc tttttctagt  961agcaactgca accggtgtac actcgagcgt acggtcgacc aagggcccat cggtcttccc 1021cctggcaccc tcctccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa 1081ggactacttc cccgaacctg tgacggtctc gtggaactca ggcgccctga ccagcggcgt 1141gcacaccttc ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac 1201cgtgccctcc agcagcttgg gcacccagac ctacatctgc aacgtgaatc acaagcccag 1261caacaccaag gtggacaaga aagttgagcc caaatcttgt gacaaaactc acacatgccc 1321accgtgccca gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc 1381caaggacacc ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag 1441ccacgaagac cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc 1501caagacaaag ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac 1561cgtcctgcac caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc 1621cctcccagcc cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca 1681ggtgtacacc ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg 1741cctggtcaaa ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc 1801ggagaacaac tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta 1861cagcaagctc accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt 1921gatgcatgag gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa 1981atgaagcttg gccgccatgg cccaacttgt ttattgcagc ttataatggt tacaaataaa 2041gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 2101tgtccaaact catcaatgta tcttatcatg tctggatcga tcgggaatta attcggcgca 2161gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga 2221aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca 2281ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca 2341ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc 2401ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc 2461catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta 2521ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctgttaaca 2581gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 2641ttaatcgcct tgcagcacat ccccccttcg ccagctggcg taatagcgaa gaggcccgca 2701ccgatcgccc ttcccaacag ttgcgtagcc tgaatggcga atggcgcctg atgcggtatt 2761ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc 2821gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 2881acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 2941cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 3001tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc 3061gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 3121cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg 3181gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 3241gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc 3301tgatgccgca tagttaagcc aactccgcta tcgctacgtg actgggtcat ggctgcgccc 3361cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 3421tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 3481ccgaaacgcg cgaggcagta ttcttgaaga cgaaagggcc tcgtgatacg cctattttta 3541taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 3601gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 3661agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 3721catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 3781ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 3841atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 3901ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgatgacgcc 3961gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 4021ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 4081ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 4141gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 4201ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc agcagcaatg 4261gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 4321ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 4381gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 4441gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 4501caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 4561cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 4621ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 4681taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 4741tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 4801gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 4861agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 4921aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 4981gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 5041gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 5101tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 5161agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 5221cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 5281gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 5341gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 5401ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 5461cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata 5521cgcaaaccgc ctctccccgc gcgttggccg attcattaat ccagctggca cgacaggttt 5581cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttacct cactcattag 5641gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga 5701taacaatttc acacaggaaa cagctatgac catgattacg aattaa GenBank ® FJ475056MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRDDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNR QIAEIGASLIKHW   1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca  61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt 721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta 781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac 841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc 901 acctcggttc tatcgattga attccaccat gggatggtca tgtatcatcc tttttctagt 961 agcaactgca accggtgtac actcgagcgt acggtggctg caccatctgt cttcatcttc1021 ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac1081 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac1141 tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc1201 ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat1261 cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta gaagcttggc1321 cgccatggcc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca1381 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca1441 tcaatgtatc ttatcatgtc tggatcgatc gggaattaat tcggcgcagc accatggcct1501 gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg1561 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg1621 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca1681 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact1741 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta1801 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag1861 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tgttaacagc ttggcactgg1921 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg1981 cagcacatcc ccccttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt2041 cccaacagtt gcgtagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc2101 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg2161 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc2221 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc2281 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct2341 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac2401 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac2461 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat2521 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa2581 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata2641 gttaagccaa ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca2701 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct2761 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg2821 aggcagtatt cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc2881 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc2941 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc3001 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc3061 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg3121 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat3181 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc3241 acttttaaag ttctgctatg tggcgcggta ttatcccgtg atgacgccgg gcaagagcaa3301 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa3361 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt3421 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct3481 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat3541 gaagccatac caaacgacga gcgtgacacc acgatgccag cagcaatggc aacaacgttg3601 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg3661 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt3721 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg3781 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg3841 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg3901 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa3961 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt4021 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt4081 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt4141 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag4201 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta4261 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat4321 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg4381 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg4441 agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag aaaggcggac4501 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga4561 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt4621 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta4681 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat4741 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg4801 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct4861 ctccccgcgc gttggccgat tcattaatcc agctggcacg acaggtttcc cgactggaaa4921 gcgggcagtg agcgcaacgc aattaatgtg agttacctca ctcattaggc accccaggct4981 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac5041 acaggaaaca gctatgacca tgattacgaa ttaa

USE OF ANTIBODIES

Antibodies described herein can be used in any method that antibodiesproduced by other means cane be used. Thus, they can be used in passivetherapy and diagnosis. Passive antibody immunization can provide a stateof immediate immunity that can last for weeks and possibly months. Somehuman IgG isotypes have serum half-lives in excess of 30 days, whichwould confer long-lived protection to passively immunized persons. Whereactive vaccines are available, they may be administered together withantibodies to both immediate and long-lasting protection. In addition,the antibodies can be administered in conjunction with one or moretherapeutic drugs for treatment or prevention of infection or fortreatment of infection. Administration of antibodies produced asdescribed herein will follow the general protocols for passiveimmunization. Antibodies for administration be prepare in a formulationsuitable for administration to a host. Aqueous compositions comprise aneffective amount of an antibody dispersed in a pharmaceuticallyacceptable carrier and/or aqueous medium. The phrases “pharmaceuticallyand/or pharmacologically acceptable” refer to compositions that do notproduce an adverse, allergic and/or other untoward reaction whenadministered to an animal, and specifically to humans, as appropriate.

As used herein, “pharmaceutically acceptable carrier” includes anysolvents, dispersion media, coatings, antibacterial and/or antifungalagents, isotonic and/or absorption delaying agents and the like. The useof such media or agents for pharmaceutical active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. For administration to humans,preparations should meet sterility, pyrogenicity, general safety and/orpurity standards as required by FDA Office of Biologics standards.

Antibodies will generally be formulated for parenteral administration,e.g., formulated for injection via the intravenous, intramuscular,sub-cutaneous, intralesional, or even intraperitoneal routes. Uponformulation, solutions will be administered in a manner compatible withthe dosage formulation or in such amount as is therapeuticallyeffective. Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject.

REFERENCES

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2. Garten, R. J., et al. Antigenic and genetic characteristics ofswine-origin 2009 A(H1N1) influenza viruses circulating in humans.Science 325, 197-201 (2009).

3. Webby, R. J. & Webster, R. G. Are we ready for pandemic influenza?Science 302, 1519-1522 (2003).

4. Yen, H. L. & Webster, R. G. Pandemic influenza as a current threat.Curr Top Microbiol Immunol 333, 3-24 (2009).

5. Palese, P. Influenza: old and new threats. Nat Med 10, S82-87 (2004).

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1. (canceled)
 2. An isolated antibody or an antigen-binding fragmentthereof, wherein the antibody comprises a light chain variable regionand a heavy chain variable region, wherein the light chain variableregion comprises the amino acid sequences of SEQ ID NOs: 3, 4, and 5 andwherein the heavy chain variable region comprises the amino acidsequences of SEQ ID NOs: 6, 7, and 8, and wherein the antibody orantigen-binding fragment specifically binds H1N1.
 3. (canceled)
 4. Theisolated antibody of claim 2 wherein the antibody is a human antibody.5. The isolated antibody of claim 2 wherein the antibody is an IgGantibody.
 6. The isolated antibody of claim 5 wherein the antibody is anIgG1 antibody.
 7. The isolated antibody of claim 6 wherein the antibodyis an IgG1, kappa antibody.
 8. The isolated antibody of claim 6 whereinthe antibody is an IgG1, lambda antibody.
 9. The isolated antibody ofclaim 2 wherein the antibody is selected from an IgM, IgA, IgD and IgEantibody.
 10. The isolated antibody of claim 1 wherein theantigen-binding fragment is selected from a Fab, a F(ab′)2 fragment, aFd fragment, an Fv fragment, and a dAb fragment.
 11. The isolatedantibody of claim 1 wherein the antibody is a scFv. 12-24. (canceled)25. The isolated antibody of claim 2 wherein the antibody has Kd forpurified H1N1 that is less than 1×10⁻⁹.
 26. (canceled)
 27. A compositioncomprising the antibody of claim 2 and a pharmaceutically acceptablecarrier.
 28. A method for reducing the risk of infection with H1N1 in ahuman subject, the method comprising administering the antibody of claim2 to the human subject thereby reducing the risk of infection of H1N1 inthe human subject.
 29. A method for treating a patient infected withH1N1 virus, the method comprising administering the antibody of claim 2to the patient infected with the H1N1 virus, thereby treating thepatient.
 29. The isolated antibody of claim 2, or the antigen bindingfragment thereof, wherein the heavy chain variable region comprises theamino acid sequence of SEQ ID NO: 2 and the light chain variable regioncomprises the amino acid sequence of SEQ ID NO:
 1. 30. The isolatedantibody of claim 2, or the antigen binding fragment thereof, whereinthe heavy chain variable region consists of the amino acid sequence ofSEQ ID NO:
 2. 31. The isolated antibody of claim 2, or antigen bindingfragment thereof, wherein the light chain variable region consists ofthe amino acid sequence of SEQ ID NO: 1.